Identification of a G 2 Arrest Domain in the E1∧E4 Protein of Human Papillomavirus Type 16

Abstract

ABSTRACT Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1∧E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1∧E4 arrests HeLa cervical epithelial cells in G 2 . 16E1∧E4 also caused a G 2 arrest in SiHa, Saos-2 and Saccharomyces pombe cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1∧E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and S. pombe cells that lack keratins, 16E1∧E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1∧E4 to be important for arrest. This region, which we have termed the “arrest domain,” contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1∧E4-mediated G 2 arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G 2 arrest was also mediated by the E1∧E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1∧E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G 2 arrest. We conclude that, like other papillomavirus proteins, 16E1∧E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.